The term "eubacteria" as used herein, refers to the group of prokaryotic organisms (bacteria) as described in, for example, Bergey's Manual of Systematic Bacteriology (N. R. Krieg and J. G. Holt, ed., 1984, Williams & Wilkins, Baltimore). As a group, the eubacteria comprise all of the bacteria which are known to cause disease in humans or animals and are of most concern with respect to detection.
The only other described group of bacteria, the archaebacteria, are biologically and genetically distinct from the eubacteria (C. R. Woese, Scientific American, 1981, Volume 244, pages 98-102). Archaebacteria as a group occupy a variety of "extreme" environments such as hot springs, strongly oxygen-depleted muds, salt brines, etc., which generally do not support the growth of eubacteria. There are no known archaebacterial pathogens and, consequently, their detection is of little clinical significance.
Eukaryotic organisms comprise the third fundamental genetic lineage which, together with the eubacteria and archaebacteria, include all known life forms (FIG. 1). Eukaryotes include humans, animals, plants and a host of organizationally less complex, free-living and parasitic "protists," including: protozoans, fungi, ciliates, etc. In a clinical context, it is particularly important that eubacteria be distinguished from eukaryotic, e.g. fungal and protozoan, infections which may present the same symptoms but require a significantly different regime of antimicrobial or chemo-therapy. These genetic distinctions thus are clinically significant from the point of view of diagnosis and antimicrobial chemotherapy.
It is an aspect of the present invention to provide nucleic acid probes which discriminate between eubacterial, human (including human mitochondrial) and fungal rRNA molecules.
It is another aspect of the present invention to provide probes and probe sets which provide a basis for discriminating between Gram positive and Gram negative eubacteria.
Methods for detecting, identifying and enumerating bacteria in normally sterile body fluids vary with the type of sample and the suspected pathogen. No currently available method is optimal for the detection of all pathogens. Often a combination of methods must be used to increase the likelihood that the pathogen will be detected. All commonly used methods for detection of, for example, bacteremia or bacterial septicemia rely on the in vitro cultivation of microbes from clinical samples. Generally, a blood sample is drawn from a patient and incubated in a rich artificial culture medium and monitored for 1 to 14 days. During this time, the medium is examined or blindly sub-cultured (plated), or assayed chemically or isotopically for evidence of bacterial growth or fermentative processes. Clinicians generally draw two or three samples of 10 milliliters of blood which may yield as few as one to ten colony forming units of bacteria for a positive diagnosis. Following the isolation of individual colonies of bacteria on diagnostic solid media and/or by Gram-staining, presumptive identification of the bacteria (or fungus) is made.
All cultivation methods suffer a number of serious shortcomings, including the following:
High material costs; PA1 Labor intensive; PA1 Technologists extensively handle dangerous bodily fluids; PA1 False positives due to handling; PA1 False negatives due to low viable cell numbers; PA1 False negatives due to fastidious media requirements of many potential pathogens; and PA1 Relatively long time to positive diagnosis and identification. PA1 a) increased sensitivity; i.e., the ability to detect said bacteria in a given sample more frequently; PA1 b) potentially significant reductions in assay cost due to the use of inexpensive reagents and reduced labor; PA1 c) accurate detection of even nutritionally fastidious strains of bacteria; PA1 d) faster results because such tests do not require the isolation of the target bacterium from the sample prior to testing; PA1 e) the ability to screen, in a batch mode, a large number of samples, and only culture those identified as "hybridization positive"; PA1 f) potential detection of phagocytized organisms eliminating the need for multiple, punctuated blood samples in order to sample the cyclical "window" of viable organisms (which probably depends on host immunological cycles); PA1 g) some reduction of technologist handling of potentially infectious body fluids; PA1 h) the ability to detect very low numbers of targets by amplifying either the bacterial signal or target using in vitro nucleic acid amplification.
Because of the relatively long time required by current methods to achieve a diagnosis and because of the potentially life threatening nature of such infections, antimicrobial therapy often is begun empirically before the results of such tests can be known.
Therefore, it is another aspect of the present invention to provide nucleic acid probes which are broadly specific for all eubacteria and which preferably do not react with other eukaryotic pathogens, especially fungi, which may be present in sampled materials.
It is yet another aspect of the present invention to provide probes which may be used in a variety of assay systems which avoid many of the disadvantages associated with traditional, multi-day culturing techniques.
It is still another aspect of the present invention to provide probes that are capable of hybridizing to the ribosomal ribonucleic acid (rRNA) of the targeted eubacterial organisms under normal assay conditions.
While Kohne et al. (Biophysical Journal 8:1104-1118, 1968) discuss one method for preparing probes to rRNA sequences, they do not provide the teaching necessary to make broad-specificity eubacterial probes.
Pace and Campbell (Journal of Bacteriology 107:543-547, 1971) discuss the homology of ribosomal ribonucleic acids from diverse bacterial species and a hybridization method for quantitating such homology levels. They do not identify particular nucleic acid sequences shared by bacteria, but absent in eukaryotes. Woese (Microbiological Reviews 51:221-271, 1987) describes the breadth of the eubacteria, in terms of rRNA sequence, but does not indicate sequences of interest for complete bacterial inclusivity. These references, however, fail to relieve the deficiency of Kohne's teaching with respect to eubacterial probes and, in particular, do not provide eubacterial specific probes useful in assays for detecting eubacteria in clinical or other samples.
Giovannoni et al. (Journal of Bacteriology 170:720-726, 1988) describe a number of probes which are claimed to be useful for the identification of broad groups of eubacteria, archaebacteria and eukaryotes. However, Giovannoni et al. do not disclose the probes of the present invention. Nor do they provide the teaching necessary to design such probes.
Hogan et al. (European patent publication WO 88/03957) describe a number of probes which are claimed to hybridize to a broad representation of eubacteria. However, Hogan et al. do not teach the probes of the present invention and also fail to relieve the deficiency of Kohne's teaching with respect to these probes.
Ribosomes are of profound importance to all organisms because they serve as the only means of translating genetic information into cellular proteins. A clear manifestation of this importance is the observation that all cells have ribosomes. Actively growing bacteria may have 20,000 or more ribosomes per cell. This makes ribosomes one of the most abundant macromolecular entities in a cell, and an attractive diagnostic assay target.
Ribosomes contain three distinct RNA molecules which in Escherichia coli are referred to as 5S, 16S and 23S rRNAs. These names historically are related to the size of the RNA molecules, as determined by their sedimentation rate. In actuality, however, ribosomal RNA molecules vary in size between organisms. Nonetheless, 5S, 16S and 23S rRNA are commonly used as generic names for the homologous RNA molecules in any bacteria, and this convention will be continued herein. Discussion will be confined to 16S and 23S rRNAs.
As used herein, probe(s) refer to synthetic or biologically produced nucleic acids (DNA or RNA) which, by design or selection, contain specific nucleotide sequences that allow them to hybridize under defined predetermined stringencies, specifically (i.e., preferentially, see below--Hybridization) to target nucleic acid sequences. In addition to their hybridization properties, probes also may contain certain constituents that pertain to their proper or optimal functioning under particular assay conditions. For example, probes may be modified to improve their resistance to nuclease degradation (e.g. by end capping), to carry detection ligands (e.g. fluorescein, 32-Phosphorous, biotin, etc.), or to facilitate their capture onto a solid support (e.g., poly-deoxyadenosine "tails"). Such modifications are elaborations on the basic probe function which is its ability to usefully discriminate between target and non-target organisms in a hybridization assay.
Hybridization traditionally is understood as the process by which, under predetermined reaction conditions, two partially or completely complementary strands of nucleic acid are allowed to come together in an antiparallel fashion (one oriented 5' to 3', the other 3' to 5') to form a double-stranded nucleic acid with specific and stable hydrogen bonds. (Note that nucleic acids do have a polarity; that is, one end of a nucleic acid strand is chemically different from another. This is defined by the polarity of the chemical linkages through the asymmetric sugar moiety of the nucleotide components. The terms 5' and 3' specifically refer to the ribose sugar carbons which bear those names. Except in rare or unusual circumstances, nucleic acid strands do not associate through hydrogen bonding of the base moieties in a parallel fashion. This concept is well understood by those skilled in the art.)
The stringency of a particular set of hybridization conditions is defined by the base composition of the probe/target duplex, as well as by the level and geometry of mispairing between the two nucleic acids.
Stringency may also be governed by such reaction parameters as the concentration and type of ionic species present in the hybridization solution, the types and concentrations of denaturing agents present, and/or the temperature of hybridization. Generally, as hybridization conditions become more stringent, longer probes are preferred if stable hybrids are to be formed. As a corollary, the stringency of the conditions under which a hybridization is to take place (e.g., based on the type of assay to be performed) will dictate certain characteristics of the preferred probes to be employed. Such relationships are well understood and can be readily manipulated by those skilled in the art.
As a general matter, dependent upon probe length, such persons understand stringent conditions to mean approximately 35.degree. C.-65.degree. C. in a salt solution of approximately 0.9 molar.